Christine Knabe

Evaluation of calcium phosphates and experimental calcium phosphate bone cements using osteogenic cultures

In this study, rat bone marrow cells (RBM) were used to evaluate two biodegradable calcium phosphate bone cements and bioactive calcium phosphate ceramics. The substances investigated were: two novel calcium phosphate cements, Biocement F and Biocement H, tricalcium phosphate (TCP), surface-modified alpha-tricalcium phosphate [TCP (s)] and a rapid resorbable calcium phosphate ceramic consisting of CaKPO(4) (sample code R5). RBM cells were cultured on disc-shaped test substrates for 14 days. The culture medium was changed daily and also examined for calcium, phosphate, and potassium concentrations. Specimens were evaluated using light microscopy, and morphometry of the cell-covered substrate surface, scanning electron microscopy, and energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. Areas of mineralization were identified by tetracyline labeling. Except for R 5, rat bone-marrow cells attached and grew on all substrate surfaces. Of the different calcium phosphate materials tested, TCP and TCP (s) facilitated osteoblast growth and extracellular matrix elaboration to the highest degree, followed by Biocements H and F. The inhibition of cell growth encountered with R 5 seems to be related to its high phosphate and potassium ion release.

Effect of β-tricalcium phosphate particles with varying porosity on osteogenesis after sinus floor augmentation in humans

This study examines the effect of two beta-tricalcium phosphate (TCP) particulate bone grafting materials with varying porosity on bone formation and on osteogenic marker expression 6 months after sinus floor augmentation. Unilateral sinus grafting was performed in 20 patients using a combination (4:1 ratio) of beta-TCP particles with 35% porosity (TCP-C) or 65% porosity (TCP-CM) and autogenous bone chips. At implant placement cylindrical biopsies were sampled and processed for immunohistochemical analysis of resin embedded sections. Sections were stained for collagen type I (Col I), alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP). Furthermore, the area fraction of newly formed bone as well as the particle area fraction were determined histomorphometrically first, apically close to the Schneiderian membrane and second, in the center of the cylindrical biopsies. In the TCP-CM patient group a larger amount of bone formation and particle degradation was observed in the apical area and thus at the largest distance from the crestal bone compared to the TCP-C group. Good bone bonding behaviour was observed with both materials. This was accompanied by expression of ALP, Col I, BSP and OC in the newly formed bone and osteogenic mesenchym in contact with the degrading particles. Both TCP materials supported bone formation in the augmented sinus floor. Six months after implantation of both types of beta-TCP particles, bone formation and matrix mineralization was still actively progressing in the tissue surrounding the particles. Consequently, a greater porosity appears to be advantageous for enhancing bone formation and particle degradation.

Morphological evaluation of osteoblasts cultured on different calcium phosphate ceramics

The objective of these investigations was to develop an in vitro test system for evaluating novel rapidly resorbable calcium phosphate ceramics of varying composition. Rat bone marrow cells were cultured on the disc-shaped test substrates for 14 days. Five calcium phosphates were examined: R1 CaNaPO4; R1/M2, composed of CaNaPO4 and MgNaPO4; R1/2, composed of CaNaPO4 and Mg2SiO4; R1 + 9% SiO2 consisting of CaNaPO4 and 9% SiO2 (wt%) and R17, Ca2KNa(PO4)2. Two studies were performed. In study I cultures were re-fed every two to three days. In study II the medium was changed daily, and calcium and phosphate concentrations of the medium were determined daily. Specimens were prepared for light microscopy and morphometric evaluation of the cell-covered substrate area, scanning electron microscopy and energy-dispersive X-ray analysis. With all materials tested except for R1/2, an increase of cellular growth was observed after changing the medium daily. Of the different calcium phosphate ceramics tested, R17 and R1/M2 facilitated osteoblast growth and elaboration of extracellular matrix to the highest degree. The inhibition of cell growth encountered with R1 in study I and R1/2 in both studies seemed to be related to a high phosphate-ion release from these materials.

In vitro investigation of titanium and hydroxyapatite dental implant surfaces using a rat bone marrow stromal cell culture system.

In this study, rat bone marrow cells (RBM) were used to evaluate different titanium and hydroxyapatite dental implant surfaces. The implant surfaces investigated were: a titanium surface having a porous titanium plasma-sprayed coating (sample code Ti-TPS), a titanium surface with a deep profile structure (sample code Ti-DPS), an uncoated titanium substrate with a machined surface (sample code Ti-ma) and a machined titanium substrate with a porous hydroxyapatite plasma-sprayed coating (sample code Ti-HA). RBM cells were cultured on the disc-shaped test substrates for 14 days. The culture medium was changed daily and examined for calcium and phosphate concentrations. After 14 days specimens were examined by light microscopy, scanning electron microscopy, energy dispersive X-ray analysis and morphometry of the cell-covered substrate surface. All test substrates facilitated RBM growth of extracellular matrix formation. Ti-DPS and Ti-TPS to the highest degree, followed by Ti-ma and Ti-HA. Ti-DPS and Ti-TPS displayed the highest cell density and thus seem to be well suited for the endosseous portion of dental implants. RBM cells cultured on Ti-HA showed a delayed growth pattern. This may be related to its high phosphate ion release.

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.

Quantification of bone tissue regeneration employing β-tricalcium phosphate by three-dimensional non-invasive synchrotron micro-tomography — A comparative examination with histomorphometry

PURPOSE This methodical study presents a novel approach to evaluate the validity of two-dimensional histomorphometric measurements of a bone biopsy specimen after sinus floor elevation by means of high contrast, high resolution, three-dimensional and non-destructive synchrotron micro-tomography (SCT). The aim of this methodical description is to demonstrate the potential of this new approach for the evaluation of bone biopsy samples. MATERIALS AND METHODS Unilateral sinus grafting was carried out exemplarily in two patients using a combination of beta-tricalcium phosphate (beta-TCP) and autogenous bone chips. For the first patient a beta-TCP with 35% porosity and in the second with 60% porosity was used. At implant placement, 6 months after sinus grafting, a cylindrical specimen was biopsied from the augmented area. Subsequent to the histological embedding in resin the specimens were imaged using a SCT facility resulting in three-dimensional (3-D) images with approximately 4 microm spatial resolution (1.5 microm pixel size) for each patients specimen. Subsequent to the SCT acquisition, tissue sections were prepared for histomorphometric analysis. RESULTS Bone area fractions determined by two-dimensional (2-D) quantitative histomorphometry and by analysis of the corresponding 2-D slice from the SCT volume data were similar. For the first biopsy specimen (beta-TCP with 35% porosity), the bone area fractions were 53.3% and 54.9% as derived by histomorphometry and by analyzing a SCT slice, respectively. For the second biopsy specimen (beta-TCP with 60% porosity) the bone area fractions were 38.8% and 39% respectively. Although the agreement between the 2-D methods was excellent, the area fractions were somewhat higher than the volume fractions computed by 3-D image analysis on the entire SCT volume data set. The volume fractions were 48.8% (first biopsy specimen) and 36.3% (second biopsy specimen). CONCLUSION Although the agreement between the 2-D methods is excellent in terms of computing the area fractions, the structural 3-D insight which can be derived from classical 2-D methods, including histomorphometric analysis is considerably limited. This fact is emphasized by the discrepancy between the measured areas and volume fractions.

Sol–gel silica controlled release thin films for the inhibition of methicillin-resistant Staphylococcus aureus

The incidence of methicillin-resistant Staphylococcus aureus (MRSA) infection has significantly increased. Generally, the success of this bacterium as a pathogen is attributed to its ability to adhere to surfaces and remain there, under the protection of an extracellular matrix known as biofilm. To combat MRSA with regular doses of vancomycin, efforts are continuously underway to increase its effectiveness. A promising technique is to use combinational therapeutics. In vitro experiments showed that farnesol can be used as an adjuvant with conventional antibiotics. Farnesol is a natural sesquiterpenoid and quorum-sensing molecule. The biggest obstacle to using this concept is that farnesol is highly water insoluble. This compromises its bioavailability if it were to be used along with vancomycin at the site of infection when the treatment needs to be administered in vivo. Herein we designed an efficient therapeutic strategy for the simultaneous delivery of both antibiotic and adjuvant in order to treat MRSA infections. We demonstrate that sufficient quantities of both vancomycin and farnesol can be incorporated into sol-gel silica applied as thin films on an implant surface. The incorporation of the hydrophobic farnesol does not affect the stability of the thin films and neither does it affect the controlled release of vancomycin. The data demonstrate the potent adjuvant effect of farnesol on vancomycin in inhibiting MRSA infection. In vitro experiments show the complete inhibition (10(6) fold reduction in growth compared to control) of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) when the ratio of vancomycin to farnesol in the sol-gel silica films is optimized. The local delivery of antibiotics minimizes the need for systemic antibiotics. The incorporation of vancomycin and farnesol into thin sol-gel films represents a new treatment paradigm for the topical delivery of antibiotics with adjuvant. The potential clinical benefits are significant and include avoiding the need for revision surgery, preventing surgical site infection and controlling healthcare costs.

Histologic evaluation of a natural permanent percutaneous structure and clinical percutaneous devices.

The longevity of percutaneous devices (PD) is often hampered by exit-site infection. The babyrussa tusk, the only permanent natural percutaneous structure, was histologically studied and the implant-tissue interface of 11 continuous ambulatory peritoneal dialysis (CAPD) catheters that had been implanted in humans was evaluated histologically. Attachment of the epidermis to the tusk surface was observed. All the CAPD catheters showed varying degrees of epidermal downgrowth. These results demonstrate that the epidermis is capable of adhering closely to a percutaneous structure. Regarding the CAPD catheters, no steady state at the epidermis-implant interface was achieved, so the risk of infection still remains.

Biological response to micron- and nanometer-sized particles known as potential wear products from artificial hip joints: Part II: Reaction of murine macrophages to corundum particles of different size distributions.

Macrophages play a pivotal role in tissue reaction and immune response. They recognize, phagocytose particles and generate cytokines to influence local cellular reactions. Friction and wear of implant components usually generates microparticles (MP) in a size range of 1-10 mum and nanoparticles (NP) in the range of 10-1000 nm. To investigate the possible impact of MP or NP on cellular reactions, we exposed murine macrophages (RAW264.7) to corundum MP and NP. The same mass was used in both NP and MP cell culture solutions, i.e. there were more NP than MP per identical volumes of culture solution. After 4, 24, 48, 72, and 96 h aliquots of cell culture supernatants were tested for different cytokines, growth factors and nitric oxide. Macrophages were stained with MGG (May-Grünwald Giemsa), counted and morphologically characterized by scanning electron microscopy and transmission electron microscopy. Particles were attached to cell surfaces and phagocytosed within cells. Cells stimulated with particles or lipopolysaccharides for positive controls showed surface modifications indicating enhanced function. Although only marginal differences between negative controls and particle-stimulated cells were observed in respect to cytokine production, exposure to corundum particles led to a decrease in the number of vital macrophages and to an increase in the number of giant cells. Corundum NP formed micron-sized aggregates in the cell culture medium and led to the production of more giant cells than MP. Sodiumdodecylsulfate polyacrylamide gel electrophoresis of the cell culture medium with particles proved the adsorption of proteins to particles.